Porcine Small Intestinal Submucosa (SIS) as a Suitable Scaffold for the Creation of a Tissue-Engineered Urinary Conduit: Decellularization, Biomechanical and Biocompatibility Characterization Using New Approaches

Abstract

Bladder cancer (BC) is among the most common malignancies in the world and a relevant cause of cancer mortality. BC is one of the most frequent causes for bladder removal through radical cystectomy, the gold-standard treatment for localized muscle-invasive and some cases of high-risk, non-muscle-invasive bladder cancer. In order to restore urinary functionality, an autologous intestinal segment has to be used to create a urinary diversion. However, several complications are associated with bowel-tract removal, affecting patients' quality of life. The present study project aims to develop a bio-engineered material to simplify this surgical procedure, avoiding related surgical complications and improving patients' quality of life. The main novelty of such a therapeutic approach is the decellularization of a porcine small intestinal submucosa (SIS) conduit to replace the autologous intestinal segment currently used as urinary diversion after radical cystectomy, while avoiding an immune rejection. Here, we performed a preliminary evaluation of this acellular product by developing a novel decellularization process based on an environmentally friendly, mild detergent, i.e., Tergitol, to replace the recently declared toxic Triton X-100. Treatment efficacy was evaluated through histology, DNA, hydroxyproline and elastin quantification, mechanical and insufflation tests, two-photon microscopy, FTIR analysis, and cytocompatibility tests. The optimized decellularization protocol is effective in removing cells, including DNA content, from the porcine SIS, while preserving the integrity of the extracellular matrix despite an increase in stiffness. An effective sterilization protocol was found, and cytocompatibility of treated SIS was demonstrated from day 1 to day 7, during which human fibroblasts were able to increase in number and strongly organize along tissue fibres. Taken together, this in vitro study suggests that SIS is a suitable candidate for use in urinary diversions in place of autologous intestinal segments, considering the optimal results of decellularization and cell proliferation. Further efforts should be undertaken in order to improve SIS conduit patency and impermeability to realize a future viable substitute.

Keywords: biomaterial; decellularization; regenerative medicine; small intestinal submucosa; tissue engineering; urinary diversions.

Figure 1

SIS conduit after gentle mechanical removal of the two-external layers of the jejunum (A). Native (B) and decellularized (C) SIS: collagenous fibre bundles are visible through the transparent surface. (D) DNA quantification performed in native and decellularized SIS samples: with NanoDrop on the left and with Qubit on the right. Data on graphs show mean ± SD. Data analysed by t-test. **** p < 0.0001 and ** p < 0.01. Merge between phase contrast and DAPI images in case of native (E) and decellularized (F) shows absence of nuclei in the latter.

Figure 2

Histological analyses of native and decellularized SIS. H&E shows the effective nuclei removal in decellularized tissue (A). Collagen and elastic fibres are revealed by Masson’s Trichrome (B) and Weigert–van Gieson (C) stainings, respectively. Alcian Blue shows the presence of glycoproteins (D). Z-stack of max intensity of phase contrast of native and decellularized SIS (E) and representative FFT (F). SHG intensity values from z-stack measurements (G). Coherency analysis from z-stack (H). Data on graphs show mean ± SD. Data analysed by t-test. * p < 0.05. Representative intensity profile for native and decellularized samples for each step during z-stack acquisition (I).

Figure 3

(A–E): Immunofluorescence of cellular and ECM components: collagen I (red) merged with phalloidin (magenta) (A), collagen IV (B), elastin (C), laminin (D) and fibronectin (E). Hydroxyproline (F) and elastin (H) quantifications are reported. No significant difference was found after hydroxyproline quantification, whereas a significant difference was found in the elastin content. Collagen estimation is reported as percentage (G), showing no statistical difference by t-test. *** p < 0.001 and ns p ≥ 0.05. The comparison between native and decellularized SIS of the ratio (R) between the peaks related to amide I and II is reported. (No significant difference was found using a t-test, p ≥ 0.05) (I). FTIR spectra are reported, showing the transmittance (%) over the wavenumbers (cm⁻¹). Dashed lines highlight peaks at 1631 cm⁻¹ (amide I and triple helix), 1558 cm⁻¹ (amide II), 1451 cm⁻¹ (all types of collagen), 1339 cm⁻¹ (collagen I and IV), 1205 cm⁻¹ (collagen IV and V), 1080 cm⁻¹ (collagen V and VI), and 1035 cm⁻¹ (all types of collagen) cm⁻¹ (J).

Figure 4

(A) Stress/strain curves of native and decellularized SIS along circumferential and longitudinal directions. Thickness, Young’s modulus (E), ultimate tensile strength (UTS), and failure strain (FS) are reported correspondingly in (B–E) comparing native and decellularized tissues. After decellularization, significant differences were found as to thickness, E and UTS, using a t-test. No significant (ns) differences were found as to FS values for both directions. Data on graphs show mean ± SD. Data analysed by t-test. **** p < 0.0001, ** p < 0.01 (n = 12) and * p < 0.05. (F) Burst pressure (cmH₂O) graph over volume (mL) in native and decellularized SIS. Burst pressure (G) and conduit length (H) values are reported. Data show mean ± SD (n = 5). Data analysed by t-test. *p < 0.05.

Figure 5

Cell proliferation on decellularized SIS. Patches were seeded with 20,000 cells/cm² and stained with phalloidin (magenta) and DAPI (cyan) (A, first row). Control group of cells seeded on plastic is reported in the second row (A). Throughout the time course, a progressive increase in cell number was found with a concurrent organization of cells along the principal directions of collagen fibres (here, z-stacks are reported of epifluorescence images). Live/dead staining on seeded SIS at days 1, 3, and 7 (B). Nuclei were stained with Hoechst in blue, live cells were stained green with calcein AM. and dead cells were stained in red by ethidium homodimer-1. For all the time points, few dead cells were detected, while an increase in the number of live cells was evident over time, showing also an increase in cell alignment along the SIS fibres. Optical density (OD) on tissue seeded with an initial number of 20,000 cells/cm² is reported (C), showing a significant difference between day 1 and day 7. Dunnett’s multiple comparisons test was performed (n = 3), comparing day 3 and day 7 to day 1 (control group). Data show mean± SD, *** p < 0.001.